1. Field of the Invention
This invention relates to immunoassay for an analyte, and more particularly relates to membrane immunoassay and particular reagents useful therein.
2. Background Information
Assay systems which are both rapid and sensitive have been developed to determine the concentration of a substance, generally referred to as the analyte, present in low concentration in a fluid sample. Immunoassays depend on the binding of an antigen or hapten to a specific antibody and have been particularly useful because they give high levels of specificity and sensitivity. These assays employ one of the above reagents in labeled form, the labeled reagent being referred to as the tracer.
Enzymes have often been used as labels in immunoassay. In conventional enzyme immunoassay (EIA), the enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a signal which is detected and measured. The signal may be a color change, detected with the naked eye or by a spectrophotometric technique, or may be conversion of the substrate to a product detected by fluorescence.
A convenient format for EIA is solid phase immunoassay in which one of the assay reagents is immobilized on a solid support. The solid support may be in the form of a dipstick, the inside wall of a test tube or cuvette or the well of a microtiter plate. A particularly useful solid support is a microporous membrane.
Membrane immunoassay is often referred to as flow-through assay. Examples of flow-through EIA wherein flow is generated by capillary action are the Bagshaw, U.S. Pat. No. 4,246,339 to Cole et al. and U.S. Pat. No. 4,632,901 to Valkirs et al. U.S. Pat. No. 4,277,560 to Gray and U.S. Pat. No. 4,812,293 to McLaurin et al. are examples of flow-through assays using pressure and vacuum respectively.
In membrane EIA, any number of liquids may be caused to flow-through the membrane to effect binding, separation and washing of assay components. The final step in most membrane EIA procedures is passage of a color developing reagent, such as a chromogen, through the membrane. The chromogen reacts with enzyme captured on the membrane to produce a color change which may be detected as evidence of the presence of analyte or measured as evidence of the concentration of analyte.
An enzyme commonly used in immunosassay is alkaline phosphatase (AP). This enzyme is present in practically all cells, and has as its principle function the removal of phosphate groups. It has been extensively studied, and is well-known to be deactivated at low pH (Schlesinger et al., Journal of Biological Chemistry, 240, 4284 (1965); McComb et al., Alkaline Phosphatase, Plenum Publ., New York, N.Y., 1979, p 413).
A problem often encountered in colorimetric assays using AP as the label results from the ubiquitous nature of the enzyme. Most assays for an antigen in a clinical sample are performed without isolation of the antigen. If AP is present in the sample (hereinafter referred to as endogenous AP), it may undergo nonspecific binding to the membrane or one of the assay components, or may not be completely removed by wash steps in the assay protocol. In such a case, positive signals from clinically negative samples may result. The present invention is directed to overcoming this problem.